11beta, 12beta-oxido-14alpha-hydroxy-delta4-pregnenes



United States Patent This invention relates to the synthesis of valuable steroids and has for its objects the provision of (a) new steroids which are physiologically active, and (b) an advantageous process for preparing the same.

The process of this invention initially comprises subjecting a steroid of the general formula wherein the 1,2-position is saturated or double-bonded to the action of the microorganism Wojnowicia gramz'nis and recovering the steroids of the general formula CH Z wherein the 1,2-position is saturated or double-bonded and Z is hydrogen or hydrox obtained thereby.

If desired these steroids, wherein Z is hydroXy, can then be esterified by treatment with the acyl halide or acid anhydride of the desired acid to yield ester derivatives of this invention. Although any acyl halide or acid anhydride may be used, the preferred esterifying reagents are those of hydrocarbon carboxylic acids of less than twelve carbon atoms, as exemplified by the lower alkanoic acids (e.g., acetic, propionic, butyric, pentanoic, hexanoic and octanoic acid), the monocyclic aromatic carboxylic acids (e.g., benzoic and p-toluic acid), the monocyclic aromatic lower alkanoic acids (e.g., phenacetic and B-phenylpropionic acid), the monocyclic cycloalkanecarboxylic acids, the monocyclic cycloalkenecarboxylic acids and the lower alkenoic acids. The reaction is preferably conducted in the presence of an organic base, such as pyridine, and results in the preparation of the corresponding 21-mono ester of the starting steroid reactant.

The final products of this invention can, therefore, be represented by the general formula wherein the 1,2-position is saturated or double-bonded, Z is hydrogen, hydroxy or the acyloxy radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms. These final products, wherein Z is hydroxy or acyloxy, are physiologically active substances which possess mineralocorticoid activity and hence can be used in lieu of known mineralocorticoids in the treatment of conditions requiring such active substances. These final products, wherein Z is hydrogen, are physiologically active substances which possess progestational activity and hence can be used in lieu of known progestational agents, such as progesterone, in the treatment of conditions requiring such active substances.

The following examples illustrate the invention (all temperatures being in centigrade):

EXAMPLE 1 1 1 3,1ZQ-Oxido-A -pregnadiene-3,20-dione To a solution of 1.0 g. of 1lfl,l2fl-oxidoprogesterone in 50 ml. of purified dioxane, 700 mg. of 2,3-dichloro-5,6 dicyanobenzoquinone is added and the resulting solution is refluxed under nitrogen for six hours. After cooling the precipitated 2,3-dichloro-5,d-dicyanohydroquinone is filtered and washed with dioxane. The combined filtrate and washings are diluted with an equal volume of chloroform and adsorbed onto 4-0 g. of Woelm neutral alumina. Elution with chloroform and crystallization of the residue of the eluates from acetone-hexane gives 11 3, 1ZB-oxido-A -preguadiene-3,20-dione.

EXAMPLE 2 J 1 8,1Zfi-oxido-Ma-hydroxyaesoxycorticosterone and 1 1,8,1 2,8-0xfd0-1 4 a-hya'roxy progesterone Surface growth from each of two three-week old agar slant cultures of Wojnowicia gramz'nis NRRL 2472, the slant containing as a nutrient medium (A): glucose, 10 g.; Difco yeast extract, 2.5 g.; K HPO 1 g.; agar, g.; and distilled water to 1 1.; are suspended in 2.5 ml. of an 0.01% sodium lauryl sulfate aqueous solution. One milliliter portions of the suspension are used to inoculate four 250 ml. conical flasks, each containing 50 ml. of the following sterilized nutrient medium (B): dextrose, 10 g. cornsteep liquor, 6 g.; NH H PO 3 g.; Difco yeast extract, 2.5 g.; Cacl 2.5 g.; and distilled water to 1 1. After four days incubation at with continuous rotary agitation (280 cycles per minute, 2 inch radius), 10% (vol/vol.) transfers are made to twenty 259 ml. conical flasks each containing m1. of fresh sterilized medium B. After an additional incubation period of three days, 10% transfers from these flasks are made to two hundred 250 ml. conical flasks each containing 50 ml. of fresh sterilized medium B. The steroid is added by adding to each flask 0.25 ml. of a sterile solution of 115,12fi-oxidoprogesterone in N,N-dirnethylformamide (6O nag/ml.) so that the medium is supplemented with 300 ig/ml. of steroid. After 48 hours of further incubation, the contents of the flasks are pooled and filtered through a Seitz clarifying pad. The flasks, mycelium and pad are washed with successive 50 ml. portions of warm water. The combined filtrate and washings have a volume of eleven liters. This is ex tracted with three 3.3 1. portions of chloroform which are combined, washed twice with 5 1. portions of water and evaporated to dryness in vacuo. The residue (2.3 g.) on crystallization from acetonehexane gives a precipitate of 115,1213- oxidodesoxycorticosterone and an ace tone-hexane mother liquor. The mother liquor is evap orated to dryness in vacuo to yield a mother liquor residue which is dissolved in benzene and chromatographed on Florisil (trademark of the Floridin Co., Tallahassee, Florida, for a magnesium silicate). Elution with chlorom... 2.86, 5.88, 6.02, 6.19,. Analysis.--Found: C, 70.15; H, 7.98.

' EXAMPLE 3 Following the procedure of Example 2, but substitutingllfl,lZfl-oxido-A -pregnadiene-3,20-dione for the 1113, 12B-oxidoprogesterone in the fermentation there is obtained 11,8,125 oxido-A -pregnadiene-14oz21-diol-3,20 dione and 115,125 oxido-A -pregnadiene-14a-ol-3,20 dione.

EXAMPLE 4 11B,12,6-oxido-J4u-hydr0xydesoxycorticosterone 21 -acetate A solution of 100 mg. of 11 3,12,6-oxido-14a-hydroxydesoxycorticosterone in 3 ml. of dry pyridine and 1 ml. of acetic anhydride is left at room temperature for 16 hours, then diluted with water and extracted with chloroform. The chloroform is washed successively with 2 N HCl, NaHCO and water and then evaporated to dryness in vacuo. Crystallization of the residue from acetone-hexane gives 11,8,12/3-oxido-14a-hydroxydesoxycorticosterone 21-acetate having M.P. about 208-210"; 113

my, 238 m,u(6=17,200); A232 2.92, 5.72, 5.82, 6.03, 6.21..

Analysis.-Calcd. for C H O (402.47): C, 68.63; H, 7.51. Found: C, 68.60; H, 7.46.

V 4 In a similar manner, by substituting other acylating agents for the acetic anhydride in the procedure of EX- ample 4, corresponding ester derivatives are obtained.

Thus, propionic anhydride and benzoyl chloride yield their respective 'propionate and benzoate ester derivatives. The invention maybe variously otherwise embodied within, the scope of the appended claims.

What is claimed is: l. A compound selected from the group consisting of steroids of the formula a .r lj t and the 1,2-dehydro derivatives'thereof, wherein Z is selected from the group consisting of hydrogen, hydroxy and the acyloxy radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms.

2. 11,8,12B-oXido-14u-hydroxydesoxycorticosterone.

3. L15,1ZB-oxido-14ot-hydroxydesoxycorticosterone 21- acetate.

4. 115,l2fl-oxido-14a-hydroxyprogesterone.

References Cited by the Examiner UNITED STATES PATENTS 2,748,149

5/56 Reichstein 260-3971 2,756,179 7/56 Fried et a1. 195-5l 7 2,830,935 4/58 Shull '195-51 2,922,798 1/ Wettstein et a1 260-39745 2,930,791 3/60 Meister et a1 260-23957 2,932,639 4/60 Oliveto et al. 260-239.55 2,951,840 9/60 Ringold et a1. 260239.55

LEWIS GOTTS, Primary Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF STEROIDS OF THE FORMULA 